In-Silico PCR

[Tongyao Wang]

Hello,

I am trying to use command to run the UCSC In-Silico PCR to check primers. And I have got /hg38/kent directory, which shows "

This directory contains the entire source tree for the

UCSC Genome Bioinformatics Group's suite of biological analysis

and web display programs as well as some of Jim Kent's own tools."

in the README file.

But I cannot find In-Silico PCR in this directory. Do you mind to help me out with this?

Thanks,

Tongyao

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Tongyao Wang

Bioinformatics

College of Science

Northeastern University

wan...@husky.neu.edu

860-617-0735

[Tongyao Wang]

Hello,

I have found the isPcr and installed it. But I am not sure how to use it, like how to give the query file and sequence file. I am really appreciate it if you can give an example.

Thanks, 

Tongyao

[Matthew Speir]

Hi Tongyao,

Thank you for your questions about the command line version of isPcr. If your run the isPcr program without any arguments, it will display the usage statement. This usage statement contains the basic information that you need to run the isPcr program such as a description of how to format your input and the various input options. Here is the usage statement:

    isPcr - Standalone v 35x1 In-Situ PCR Program
    usage:
       isPcr database query output
    where database is a fasta, nib, or twoBit file or a text file containing
    a list of these files,  query is a text file file containing three columns: name,
    forward primer, and reverse primer,  and output is where the results go.
    The names 'stdin' and 'stdout' can be used as file names to make using the
    program in pipes easier.
    options:
       -ooc=N.ooc  Use overused tile file N.ooc.  N should correspond to
                   the tileSize
       -tileSize=N the size of match that triggers an alignment. 
               Default is 11 .
       -stepSize=N spacing between tiles. Default is 5.
       -maxSize=N - Maximum size of PCR product (default 4000)
       -minSize=N - Minimum size of PCR product (default 0)
       -minPerfect=N - Minimum size of perfect match at 3' end of primer (default 15)
       -minGood=N - Minimum size where there must be 2 matches for each mismatch (default 15)
       -mask=type  Mask out repeats.  Alignments won't be started in masked region
                   but may extend through it in nucleotide searches.  Masked areas
                   are ignored entirely in protein or translated searches. Types are
                     lower - mask out lower cased sequence
                     upper - mask out upper cased sequence
                     out   - mask according to database.out RepeatMasker .out file
                     file.out - mask database according to RepeatMasker file.out
       -makeOoc=N.ooc Make overused tile file. Database needs to be complete genome.
       -repMatch=N sets the number of repetitions of a tile allowed before
                   it is marked as overused.  Typically this is 256 for tileSize
                   12, 1024 for tile size 11, 4096 for tile size 10.
                   Default is 1024.  Only comes into play with makeOoc
       -flipReverse Reverse complement reverse (second) primer before using
       -out=XXX - Output format.  Either
          fa - fasta with position, primers in header (default)
          bed - tab delimited format. Fields: chrom/start/end/name/score/strand
          psl - blat format.

I hope this is helpful. If you have any further questions, please reply to gen...@soe.ucsc.edu. All messages sent to that address are archived on a publicly-accessible Google Groups forum. If your question includes sensitive data, you may send it instead to genom...@soe.ucsc.edu.

Matthew Speir
UCSC Genome Bioinformatics Group

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[Tongyao Wang]

Thanks for your help, Matthew. Here I have another question. I have the files for each chromosome sequence from ucsc web. Now I want to check the primers on every chromosome, so I need to combine them to one fasta file?

Thanks,

Tongyao

[Luvina Guruvadoo]

Hello Tongyao,

Please note this part of the usage statement:

...where database is a fasta, nib, or twoBit file
or a text file containing a list of these files...

"database" can be a list of your fasta files. Alternatively, you could write a short script and run isPcr from the commandline against each chromN.fa with chromN.out as output. After all the chroms have been run, you can concatenate the outputs together.

I hope this helps. If you have any further questions, please reply to gen...@soe.ucsc.edu. All messages sent to that address are archived on a publicly-accessible forum. If your question includes sensitive data, you may send it instead to genom...@soe.ucsc.edu.

- - -
Luvina Guruvadoo
UCSC Genome Bioinformatics Group

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